Cloning of Atlantic halibut growth hormone receptor genes and quantitative gene expression during metamorphosis

Abstract

To gain insight into the possible regulatory role of the growth hormone (GH)-insulin-like growth factor I (IGF-I) system in flatfish metamorphosis, body GHR gene expression as well as IGF-I protein content was quantified in larval Atlantic halibut throughout metamorphosis (developmental stages 5–10). The cDNA of the full-length GH receptor (hhGHR) was cloned from adult liver and characterized. The hhGHR shows common features of a GHR, including a (Y/F)GEFS motif in the extracellular domain, a single transmembrane region, and an intracellular domain containing a Box 1 and Box 2. Additionally, a truncated GHR (hhGHRtr), similar to turbot and Japanese flounder GHRtr, was cloned and sequenced. These sequences are highly similar to the full-length and truncated GHRs in turbot (89%/86%) and Japanese flounder (93%/91%) with lower identity with other fish type I GHR (⩽81%) and type II GHRs (⩽58%). A quantitative real-time RT-PCR assay was used to measure hhGHR and hhGHRtr mRNA content in normally and abnormally metamorphosed individuals at six developmental stages, from early pre-metamorphosis to post-metamorphosis, when the fish is considered a juvenile. The level of hhGHR gene expression was highest at pre-metamorphic stage 6 and at stage 8 at the onset of metamorphosis, and then decreased during metamorphic climax and post-metamorphosis. Expression of hhGHRtr reached highest levels at stage 6 and then decreased to post-metamorphosis. The ratio of expression between the full-length and the truncated GHR (hhGHR:hhGHRtr) varied among stages and was highest at the onset of metamorphosis and at metamorphic climax. A radioimmunoassay was used to measure halibut IGF-I body content throughout metamorphosis. IGF-I increases from early metamorphosis to the onset of metamorphosis and then decreases towards post-metamorphosis. In comparison between normally and abnormally metamorphosing larvae, IGF-I content, hhGHR and hhGHRtr mRNA levels were reduced in the abnormal fish. These data indicate that the GH-IGF-I system either has a regulatory role in metamorphosis, or is being affected as a consequence of the abnormal metamorphosis.

Introduction

Growth hormone (GH) has multiple functions in fish, regulating skeletal and soft tissue growth and metabolism as well as osmoregulation, reproduction and immune response (see Reinecke et al., 2005). GH regulates physiological functions either by acting directly on target tissue or by stimulating the hepatic production of insulin-like growth factor I (IGF-I). IGF-I in turn stimulates tissue and skeletal growth (see Kopchick and Andry, 2000). GH mediates its biological effects by binding to membrane receptors on target tissues, initiating intracellular signaling pathways. GH receptor (GHR) mRNA is expressed in many fish tissues, including liver, muscle, fat, bone, kidney, brain, pancreas, spleen, gall bladder, ovary, testis, esophagus, stomach, intestine, heart and gills (Calduch-Giner et al., 2001, Fukada et al., 2004, Kajimura et al., 2004, Lee et al., 2001, Nakao et al., 2004, Tse et al., 2003, Very et al., 2005), further underlining the pluripotency of GH. A second form of the GHR with a truncated intracellular domain is found in two flatfish species, turbot (Scophthalmus maximus, Calduch-Giner et al., 2001) and Japanese flounder (Paralichtys olivaceus, Nakao et al., 2004), as well as in some mammalian species, including human, rabbit, rat and mouse (see Edens and Talamantes, 1998). This receptor has been suggested to act as a dominant–negative inhibitor of the full-length receptor and to increase levels of GH binding protein (Calduch-Giner et al., 2001). GHRs have been sequenced in over 20 teleost species, but knowledge of the physiological significance of GHR gene expression during fish development is limited.

Flatfish metamorphosis is a dynamic process involving tissue remodeling and differentiation in addition to a variety of biochemical and physiological alterations mediated by differential gene expression and endocrine regulation. Thyroid hormones, cortisol, prolactin and sex steroids have been shown to influence metamorphosis in the Japanese flounder (see de Jesus et al., 1993, Yamano and Miwa, 1998), but little is known about the potential physiological role(s) of the GH-IGF-I system in this process. Although ovine GH does not affect in vitro or in vivo fin ray shortening in Japanese flounder metamorphosis, pituitary GH mRNA increases throughout metamorphosis (de Jesus et al., 1994). GH is sometimes indicated to act as a juvenile hormone or antimetamorphic in amphibian metamorphosis (Shintani et al., 2002, Takada and Kasai, 2003), but the data are inconclusive (Bern et al., 1967, Huang and Brown, 2000, Wright et al., 1994). GH-containing somatotrophs are found from an early premetamorphic stage in Atlantic halibut (Einarsdóttir et al., 2006a), and the pituitary GH content increases in proportion to size in metamorphosing larvae (Einarsdóttir et al., 2006b) This, together with the multifunctional role of GH in fish, which is to a high degree closely linked to growth and developmental processes such as salmon smoltification (see Björnsson, 1997), makes it likely that the GH-IGF-I system plays a physiological role during Atlantic halibut metamorphosis.

Small larval size precludes studies of circulating hormone in fish. Therefore, although GH is present in the pituitary gland of larval halibut from early pre-metamorphic stages (Einarsdóttir et al., 2006a, Einarsdóttir et al., 2006b), it is unknown if the pituitary GH is being secreted into the circulation during larval development and metamorphosis. In addition to hormone secretion, a critical prerequisite for the functionality of any endocrine system is the presence of receptors in target tissues.

While the question of GH secretion cannot be resolved, the aim of this study was to examine if GH receptors are present in tissues, and thus to clarify if GH could exert endocrine control during halibut metamorphosis. As a component of the GH-IGF-I system, IGF-I production is down-stream from GH activation of its receptor. Thus, assessment of IGF-I could give additional information about the activity of the GH-IGF-I system during metamorphosis. To reach these aims, a quantitative measure of GHR expression in halibut was established by cloning both the full-length and the truncated GHR forms. Their expression, as well as body IGF-I protein content, was studied throughout the metamorphic process.

Further, it was hypothesized that if the GH-IGF-I system is of importance for the halibut metamorphic process, GHR expression and IGF-I content will differ between larvae going through normal metamorphosis and larvae going through abnormal/incomplete metamorphosis. To reach this aim, each sampled larva was photographed, staged according to Saele et al., 2004, and classified as normal or abnormal.